In patients with acute lung injury and pulmonary edema, alveolar gas exchange is impaired which may result in significant hypoxemia. In the previous cycle of this grant proposal we have determined that hypoxia impairs the lung's ability to clear edema by inhibiting the Na,K-ATPase in the alveolar epithelium. The focus of this application is to determine the mechanisms regulating the effects of severe hypoxia of 1.5%, 3% or 5% (~10 to 40 mm Hg) on alveolar epithelial function focusing on the regulation of Na,K-ATPase endocytosis and degradation. We will determine whether a brief exposure of alveolar epithelial cells (AEC) to hypoxia results in mitochondrial reactive oxygen species mediated phosphorylation of the AMP Kinase (AMPK) leading to activation of protein kinase C zeta (PKC?) and the endocytosis/degradation of Na,K-ATPase. We have previously reported that in AEC exposed to hypoxia the plasma membrane Na,K-ATPase was degraded relatively rapidly, while the degradation of the intracellular Na,K-ATPase molecules was much slower. We reason that this apparent discrepancy is related to the "acute versus chronic" effects of hypoxia and cell adaptation involving the hypoxia inducible factor (HIF1() during chronic hypoxia. Thus, we propose to dissect the mechanisms that regulate the effects of acute and prolonged hypoxia. We reason that prolonged hypoxia exposure results in cell adaptation via a HIF1( mediated mechanism which leads to downregulation of the PKC? protein and thus prevents further endocytosis/degradation of the Na,K-ATPase. As such, we will study the effects of hypoxia on the alveolar epithelium by focusing on the mechanisms of Na,K-ATPase regulation via three interrelated aims: in Specific Aim 1 we propose to determine whether hypoxia activates AMPK and its role in the regulation of alveolar epithelial Na,K-ATPase and fluid reabsorption; in Specific Aim 2 we will study whether the HIF ubiquitin ligase, von Hippel Landau protein (pVHL), regulates Na,K-ATPase endocytosis/degradation during hypoxia, and in Specific Aim 3 we will determine whether HIF1( stabilization leads to PKC? ubiquitination and degradation as a mechanism of regulating total cell Na,K-ATPase levels and thus, alveolar epithelial function. Experiments have been conducted for each of the specific aims and the preliminary results support the feasibility of this proposal. Completion of the proposed studies will provide novel information on the effects of hypoxia on the alveolar epithelium, specifically as it pertains to mechanisms of inhibition of the Na,K-ATPase, impairment of alveolar epithelial function and cell adaptation to hypoxia which may be of importance for the understanding and design of novel approaches to improve alveolar epithelial function in patients with pulmonary edema. PROJECT NARRATIVE: Acute respiratory distress syndrome (ARDS) has a high mortality rate of ~40 percent or ~60,000 annually. Damage to pulmonary capillaries can cause a leak leading to flooding of the airspaces or pulmonary edema. This grant proposal focuses on how the alveolar epithelium responds and adapts to low levels of oxygen which is common in patients with ARDS or during ascent to high altitudes. The information generated from these experiments will provide new insights to be used in the treatment of patients with ARDS. [unreadable] [unreadable] [unreadable]